The present invention relates to isolation of cytotoxic T lymphocyte (CTL) clones. The CTL clones of the present invention have been isolated by successive steps of stimulation and testing of lymphocytes with antigen presenting cells which present antigens derived from different expression systems, e.g., from recombinant Yersinia, recombinant Salmonella, or recombinant viruses. The present invention further relates to isolated CTL clones that are specific for proteins of the MAGE family. Antigenic peptides as well as the peptide/HLA complexes which are recognized by the isolated CTL clones are also provided.
An important facet of the immune response in a mammalian subject is the recognition by T cells of the complexes of the cell surface molecules, i.e., the complexes of peptides and HLA (human leukocyte antigens) or MHC (major histocompatibility complexes) molecules. These peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecules. See in this regard, Male et al., Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters 6-10. The interaction between T cell and HLA/peptide complexes is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities.
Most progressively growing neoplastic cells express potentially immunogenic tumor-associated antigens (TAAs), also called tumor rejection antigens (TRAs). A number of genes have been identified that encode tumor rejection antigen precursors (or TRAPs), which are processed into TRAs in tumor cells. Such TRAP-encoding genes include members of the MAGE family, the BAGE family, the DAGE/PRAME family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp 100, MUC-1, TAG-72, CA125, mutated proto-oncogenes such as praise, mutated tumor suppressor genes such as p53, tumor associated viral antigens such as HPV16 E7. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler et al. (1997) Advances in Pharmacology 40: 309-337, Academic Press, Inc., San Diego, Calif.
TRAs, like other antigenic epitopes, are presented at the surface of tumor cells by MHC molecules and have been shown to induce a CTL response in vivo and in vitro. See, for example, van der Bruggen et al. (1991) Science 254: 1643-1647. However, such TRA expressing tumor cells do not provoke reliable anti-tumor immune responses in vivo that are capable of controlling the growth of malignant cells. Boon et al. (1992) Cancer Surveys 13: 23-37; T. Boon (1993) Int. J. Cancer 54: 177-180; T. Boon (1992) Advances Cancer Res. 58: 177-209. Thus, generation of CTL clones that recognize specific TRAs provides a powerful tool for tumor therapeutics. The identification of TRAs also allows the design of recombinant vaccines for the treatment of various pathological conditions.
The present invention provides a novel procedure for isolating CTL clones. By following such procedure, novel CTL clones have been isolated that recognize specific antigenic peptides of proteins, preferably of the MAGE family. The MHC molecules presenting these peptides have been identified as well.
One embodiment of the present invention provides methods for isolating CTL clones from a blood sample.
The methods of the present invention include successive steps of stimulating and testing lymphocytes with antigen presenting cells. Such methods, by employing different antigen presenting cells at different steps, significantly reduce non-specific CTL activities generated in the procedure and permit more efficient isolation of CTL clones.
Antigen presenting cells which are used in the methods of the present invention can differ in cell type and/or in the expression system from which the antigen to be presented is derived. Cells which can be employed as antigen presenting cells in the present methods include professional and facultative antigen presenting cells. A preferred antigen presenting cell is an autologous dendritic cell, an autologous B cell transformed with EBV, or an activated T cell.
Antigen presenting cells can be modified by a variety of ways to effect the expression of an antigen of interest at the cell surface, preferably, by infection with a recombinant Yersinia, recombinant Salmonella, or recombinant viruses. Preferred recombinant viruses include vaccinia, canarypox virus, other pox viruses, adenovirus, herpes simplex virus, and retrovirus.
The protein against which CTL clones are generated can be a tumor associated protein, an antigenic protein of a pathogen, or the like. Preferably, the protein is a member of the MAGE family, in particular, MAGE-A1, MAGE-A3 and MAGE-A4.
In another embodiment, the present invention contemplates CTL clones isolated by using the methods of the present invention.
In a preferred embodiment, the present invention provides isolated CTL clones that are specific for peptide/HLA complexes SAYGEPRKL(SEQ ID NO: 2)/HLA-Cw3, DPARYEFLW(SEQ ID NO: 42)/HLA-B53, GVYDGREHTV(SEQ ID NO: 44)/HLA-A2, SAFPTTINF(SEQ ID NO: 47)/HLA-Cw2, EVYDGREHSA(SEQ ID NO: 48)/HLA-A28, AELVHFLLL (SEQ ID NO: 55)/HLA-B40, and RVRFFFPSL (SEQ ID NO: 57)/HLA-B7, respectively.
In a more preferred embodiment, the present invention provides isolated CTL clones LB1137 462/F3.2, LB1801 456/H7.11, LB1118 466/D3.31, LB 1801 456/H8.33, LB1137 H4.13, LB1841 526/F7.1 and LB1803 483/G8.4.
Furthermore, the present invention provides methods of identifying antigenic peptide epitopes of a protein by using CTL clones isolated following the methods of present invention.
In still another embodiment, the present invention provides newly isolated antigenic peptides, DPARYEFLW (MAGE-A1 258-266) (SEQ ID NO: 42), GVYDGREHTV (MAGE-A4 230-239) (SEQ ID NO: 44), SAFPTTINF(SEQ ID NO: 47) (MAGE-A1 62-70), EVYDGREHSA(SEQ ID NO: 48) (MAGE-A1 222-231), AELVHFLLL (SEQ ID NO: 55) (MAGE-A3 114-122), RVRFFFPSL (SEQ ID NO: 57) (MAGE-A1 289-297). Nucleic acid sequences encoding such peptides are also contemplated.
In another embodiment, the present invention provides isolated peptide/HLA complexes, peptide SAYGEPRKL (SEQ ID NO: 2) complexed with HLA-Cw3, peptide DPARYEFLW(SEQ ID NO: 42) complexed with HLA-B53, peptide GVYDGREHTV(SEQ ID NO: 44) complexed with HLA-A2, peptide SAFPTTINF(SEQ ID NO: 47) complexed with HLA-Cw2, EVYDGREHSA(SEQ ID NO: 48) complexed with HLA-A28, AELVHFLLL (SEQ ID NO: 55) complexed with HLA-B40, and RVRFFFPSL (SEQ ID NO: 57) complexed with HLA-B7.
In another embodiment, cells expressing any of these peptide/HLA complexes are contemplated.
Still another embodiment of the invention provides pharmaceutical compositions which include any one of the isolated CTL clones, the antigenic peptides, the peptide/HLA complexes, and cells expressing the peptide/HLA complexes of the present invention.
In a further aspect, the present invention provides methods useful for diagnosing and treating various pathological conditions.
One embodiment of the present invention provides methods of diagnosing in a subject, a pathological condition characterized by an abnormal expression of a peptide/HLA complex, by detecting the presence of cells abnormally expressing such complex in the subject.
Another embodiment of the present invention provides methods of detecting in a subject, the presence of cells abnormally expressing a peptide/HLA complex of the present invention by using an isolated CTL clone of the present invention which specifically recognizes such complex.
One embodiment of the present invention provides methods of diagnosing in a subject, a pathological condition characterized by an abnormal expression of a peptide/HLA complex, by detecting an increased frequency of CTL cells specific for such complex.
Another embodiment of the present invention provides methods of detecting in a subject the presence, of CTL cells specific for a peptide/HLA complex of the present invention by using an antigen presenting cell expressing such complex at the cell surface.
In still another embodiment, the present invention provides methods of treating a subject of a pathological condition characterized by an abnormal expression of a peptide/HLA complex of the present invention by administering to the subject, a therapeutically effective amount of cells of a CTL clone specific for such complex.
Another embodiment provides methods of treating a subject of a pathological condition characterized by an abnormal expression of a peptide/HLA complex of the present invention, by administering to the subject a therapeutically effective amount of the peptide.
Still another embodiment provides methods of treating a pathological condition characterized by an abnormal expression of a peptide/HLA complex of the present invention, by obtaining antigen presenting cells from the subject, modifying such cells to effect a presentation of the peptide/HLA complex at the cell surface, and then reperfusing such xe2x80x9cloadedxe2x80x9d cells into the subject.